Preparation Of Graphene Oxide Composite Nanoparticles Loading Timosaponin A-Ⅲ And Its Efficacy In Treating Melanoma

Objective: To study the therapeutic effect of Timosaponin A-Ⅲ(TSAⅢ)on melanoma B16F10 cells and animal models.In order to construct a drug-assisted photothermal combined treatment strategy for melanoma,graphene oxide(GO)was used as a carrier to load TSAⅢ.It aims to improve the bioavailability of TSAⅢ and enhance its anti-tumor efficacy,providing new strategies for new delivery systems and combined treatments of traditional Chinese medicine.Methods:(1)In vitro efficacy: To study the in vitro inhibitory effect of TSAⅢ on melanoma B16F10 cells.The CCK8 method was used to determine the effect of TSAⅢ on the survival rate of B16F10 cells;The Wound Healing Assay was used to evaluate the inhibitory effect of TSAⅢ on the migration of B16F10 cells.(2)Anti-tumor mechanism in vitro: After administration with TSAⅢ,observed the cell status under a microscope to evaluate its death mode;Detected the apoptosis of B16F10 cells by confocal microscope and flow cytometry;Evaluated the effect of TSAⅢ on the expression of B16F10 cell apoptosis and migration-related proteins by Western Blot;Detected the respiratory ability of B16F10 cells by Seahorse;Observed the changes in mitochondrial membrane potential and ROS content of B16F10 cells by confocal microscop.(3)Functional vector construction and drug loading evaluation: Added the foaming agent polyvinyl alcohol(PVA)and the pore-forming agent polyethylene glycol(PEG)to the GO aqueous solution,and prepared uniform,dense and porous graphene oxide functional drug-carrying materials(GPP);The preparation process and carrier of GPP were characterized by Fourier Transform Infrared Spectroscopy(FTIR),Scanning Electron Microscopy(SEM),Transmission Electron Microscopy(TEM)and other technologies.The GPP-TSAⅢ composite nanoparticles were prepared by loading TSAⅢ into GPP by physical adsorption,and further characterized by FTIR,SEM and TEM techniques,and the drug loading and release rate of the GPP-TSAⅢ composite nanoparticles were determined by HPLC-ELSD.(4)In vivo anti-tumor efficacy study: Established a B16F10 cell tumor-bearing mouse model,administered intratumorally,combined with in vivo imaging of small animals to evaluate the in vivo targeting of TSAⅢ and GPP-TSAⅢ composite nanoparticles;Using mouse body weight,tumor volume and tumor weight as indicators,the in vivo efficacy of TSAⅢ and GPP-TSAⅢ composite nanoparticles were compared;Pathological sections were used to evaluate the in vivo safety of TSAⅢ and nanoparticles in nude mice.Results:(1)In vitro efficacy: CCK8 method experimental results showed that TSAⅢ can significantly reduce the survival rate of B16F10 cells,inhibit cell migration and is dose-dependent;Microscopic observations showed that TSAⅢ caused the morphology of B16F10 cells to change,the cells appeared swelling,a large number of vacuoles appeared in the cytoplasm,and the membrane surface blistered.(2)Molecular mechanism of in vitro drug efficacy: Under the microscope,the nuclei of B16F10 cells were observed to shrink or appear fragmented.At the same time,it was confirmed that the cells were apoptosis by flow cytometry;The expression of apoptosis-related proteins and migration-related proteins were detected to change;Tested by Seahorse,mitochondrial basal respiration,ATP conversion capacity,respiration capacity,proton leakage,and indirectly measured ATP production levels have decreased significantly,and glycolysis levels have also decreased;The kit test found that the level of intracellular ROS increased and the mitochondrial membrane potential decreased significantly.(3)Carrier synthesis and drug loading: The results of various technical characterizations showed that the constructed GPP carrier has a porous structure,has good biosafety and hydrophilicity,and has high efficiency of photothermal conversion,high drug loading and a slow drug release trend.(4)Anti-tumor efficacy in vivo: In the B16F10 tumor-bearing mouse model,the tumor inhibition rates of the TSAⅢ free drug group and GPP-TSAⅢ+Laser group were 51.40 ± 16.61 % and 97.03 ± 1.11 %,respectively.The GPP-TSAⅢ+Laser group had the best tumor inhibition effect,and the tumor inhibition rate was 1.9 times that of the TSAⅢ monomer drug group.The results showed that the GPP-TSAⅢ+Laser group embodies the dual advantages of GPP+Laser photothermal therapy and TSAⅢanti-tumor,and achieves the combined treatment effect;The tissue section results also showed that GPP-TSAⅢ+Laser has no obvious tissue toxicity to various organs,and the biological safety is good.Conclusion: In vitro studies of this article revealed that TSAⅢ may act on cell mitochondria,destroy mitochondrial function,induce the expression of apoptosis-related proteins and migration-related proteins,thereby inhibiting the proliferation and migration of B16F10 cells;The successfully constructed GPP-TSAⅢcomposite nanoparticles have significant anti-tumor efficacy in vivo,which not only prolongs the residence time of TSAⅢ at the tumor site,but also increases the uptake rate of the tumor cells to the drug;At the same time,the drug delivery system and photothermal therapy have further improved the therapeutic effect of tumors and exerted the value of combined therapy.Therefore,the research of this article will provide an experimental basis for exploring the molecular biological mechanism of TSAⅢ treatment of melanoma and the combined treatment strategy of drugs and photothermal.